OPEN Experimental & Molecular Medicine (2017) 49, e397; doi:10.1038/emm.2017.225 Official journal of the Korean Society for Biochemistry and Molecular www.nature.com/emm

REVIEW

Are the view of pylori colonized in the oral cavity an illusion?

JKC Yee

Urea breath test (UBT), as a leading preferred non-invasive diagnostic technology, but may not be able to detect oral H. pylori. With negative results of UBT, the patient may have an oral infection. On the basis of the fact of success, eradication rate may increase by 21% in the 95% Cl range after the elimination of oral H. pylori, the author believes oral H. pylori does exist and the oral cavity is the second colonized site aside its primary site of the stomach. H. pylori migrated out of Africa along with its human host circa 60 000 years ago; they are not lives in stomach only. In this review article, evidence established in recent years studies with use more appropriate technology had been listed and discussed. The author considers the oral cavity is a black hole for H. pylori infection that significant effective on gastroenterology and another medical field. The role of the oral cavity as the source of H. pylori infection is so controvert in past years. It seems like a human being having a second-time face to discover H. pylori in the history. Experimental & Molecular Medicine (2017) 49, e397; doi:10.1038/emm.2017.225; published online 24 November 2017

INTRODUCTION because the majority of physicians and scientists in this field do Most scientists in this field proposed there are no living not consider oral H. pylori are living . I estimate ~ 20% H. pylori lives in the mouth and positive response of the oral of the population of Asia having H. pylori of oral cavity.4 There cavity by PCR due to reflux from the stomach. The survive of are ~ 280 million people of China having oral H. pylori H. pylori in the oral cavity lives for only a few hours. If this infection. There are 280 million people have the problem of proposal is right, then the dead bacteria should not have any recurrences of stomach H. pylori infection. The abuse and negative effective on eradication of stomach H. pylori infec- overuse of antibiotics occur everywhere.1,3,4 As today, antibiotic tions. However, there are a number of reports that indicated pollution that appears in food, water, even in children’surine eliminated oral H. pylori may help more patients recover from that become a serious concern.5 If we do not stop this abuse, stomach infection.1,2 These reports concluded that there is a antibiotic may kill 80 000 per year and cost 11.7 billion dollars significant relationship between stomach and oral H. pylori as medical expenses in China. This review will provide facts infection. This finding greatly explains why the annual H. pylori that indicating oral H. pylori infection exist and discuss how to recurrence rates were so high with the first year being 13.2% eliminating them without antibiotic. followed by the second-year and the third-year both being 18.4%, due to oral cavity infection.3 In developed countries, the THE CLOSE RELATIONSHIP BETWEEN PERIODONTAL H. pylori recurrence rates after successful eradication were very HEALTH AND H. PYLORI INFECTION low—which means that discrepancy exists and is likely Dye BA et al.6 report that a clinical periodontal study on 4504 determined by economic status. However, it still continues to participants during 10 years period that show the close relation be a considerable controversy on the fundamental issue of oral between the depths of advanced pockets and a positive blood H. pylori. For example, how the stomach infection occurs? test for H. pylori antibodies. They concluded that poor H. pylori stay in the mouth, then via the oral cavity come down periodontal health having periodontal pockets 45 mm always to stomach? What is the premium function of the oral cavity in associated with H. pylori infection of adults in US population.6 this process? Or the mouth is a second colonization site of Fernández-Tilapa G et al.7 use blood H. pylori antibody tests H. pylori? Hence, if the mode of H. pylori transmission remains found that the prevalence of H. pylori in oral cavity was higher unknown, we are unable to interrupt the spread of infection. (18.5%) among seropositive subjects compared with seronega- The resolution on these disputable issues is so important tive persons in México. However, they concluded there were no

Research Lab of Oral H pylori, Everett, WA, USA Correspondence: Dr JKC Yee, Research Lab of Oral H pylori, 125 130th Street SE, Everett, WA 98208, USA. E-mail: [email protected] Received 14 December 2016; revised 20 March 2017; accepted 23 March 2017 Oral H. pylori JKC Yee

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association between the presence of H. pylori and oral hygiene are not an important reservoir for H pylori and are probably habits. Furthermore, Nisha KJ et al.8 reported that H. pylori not a significant factor in transmission of the organism.19 may colonize in dental plaque that shows a strong relation with Author did not agree with their conclusion because the method periodontal diseases. Tsami A et al.9 also detected H. pylori of cultures used for high concentration of H. pylori in presenting in the subgingival dental plaque of children as well stomach not for oral cavity where the concentration of as their family. H. pylori at very low level. Author will discussion why failure Several reports have indicated that H. pylori colonies could of cell cultures occur in later section. be grown only from root canals but not from plaque. The root However, there are a number of published articles that canals of endodontic-infected teeth could be a reservoir for indicate there were no correlations between H. pylori gastritis living H. pylori that could serve as a potential source of and dental hygiene or periodontal disease. They concluded that transmission.10,11 either dental plaque or dentures have nothing to do with Recent studies have shown that they consistently found stomach H. pylori infection. At least, they consider oral H. pylori live in the oral cavity that strong like with recurrence H. pylori is not a significant factor cost stomach infection of stomach H. pylori infection. Furthermore, in a review article, and they areorganisms in transmission in the mouth. However, they indicted the treatment of periodontal disease may help the the key fact is that they did indeed detect H. pylori in the oral improvement of the symptoms of H. pylori-positive dyspeptic cavity, but with a different view of conclusion.20,21 They think patients.2 A large size of a clinical study of China shown a high the results of such studies should be considered prudently percentage of adults suffers from oral H. pylori infection, which because the oral cavity is the residence of several urease- also strong like with oral diseases; such as periodontal diseases producing species, including Streptococcus spp., Haemophilus and caries.12 spp. and Actinomyces spp. Those organisms also have high A review and Meta-analysis included 48 articles and 12 urease activity in dental plaque which is nothing to do with oral clinical trials, as well as a meta-analysis designed in 2011 that H. pylori.WhilethediagnosisofH. pylori in gastric samples you indicated a strong association between H. pylori infection of may see the microscopic appearance such as Gram-negative, mouth and H. pylori stomach infection. They found H. pylori curved or spiral-shaped rods which may not as H. pylori.Ifyou are the etiologic agent of periodontal disease. They concluded use microscopic appearance as a standard to check oral samples that there is a strong relation between mouth and stomach H. you can found many species have spirochetes appearance, pylori infection.13 Recent several meta-analyses report a similar including spp. so this standard has low specificity. conclusion regarding oral cavity and stomach.14–17 The total We should be prudent to make any conclusion.22 participants involved in the above meta-analyses are more than Namiot et al.23 conducted a clinical study. They reported in 20 000 individuals (Table 1). 65.6% of an adult having H. pylori antigens exist in dental Furthermore, combining full-mouth disinfection plus triple plaque. They concluded the occurrence of H. pylori antigens of therapy on periodontitis patients for oral H. Pylori infection dental plaque of natural teeth is not link with oral health status. increasing eradication success rate of stomach H. pylori After remove dental plaque of natural teeth and removable infection.18 dentures, the H. pylori still stay in mouth. However, Silva Although meta-analysis provided a right direction of period- et al.24 reported H. pylori was existing in the supragingival ontal diseases and H. pylori infection, however we should also plaque, but not in the subgingival plaque in the case if the discussion each individual study further since they reported a patient has periodontal disease and upper gastric diseases. They correlation in result but with negative conclusion, For example, concluded H. pylori may colonize in the supragingival site and A clinical study shows H pylori was detected in specimens of 34 it is strong like with oral hygiene. Chaudhry et al.25 suggested patients (54%). Because all of the cultures of dental plaque use two genes of the bacterium simultaneously amplified as were negative. They concluded that dental plaque or dentures compared to one gene amplification only then we have better

Table 1 Meta-analysis of periodontal disease and oral H. pylori associated with stomach H. pylori infection published during 2011–2016

Author Year Number Conclusion

Ren et al.15 2016 691 participants Periodontal therapy increased eradication rate of stomach infection Sayed et al.16 2014 4959 participants. Articles published during 1990–2012 Oral H. pylori increased stomach re-infection Adler et al.13 2014 45000 participants Close relation oral H. pylori and stomach H. pylori infection 48 articles and 12 clinical trials Marbaix et al.14 2013 4 5000 participants Close relation oral H. pylori and stomach H. pylori infection Included 48 articles Navabi et al.17 2011 1861 participants Close relation oral H. pylori and stomach H. pylori infection Zou and Li71 2011 46000 participants Close relation oral H. pylori and stomach H. pylori infection Articles published during 2010–2011

Experimental & Molecular Medicine Oral H. pylori JKC Yee

3 chance to found H. pylori in dental plaque which may be a effective on systemic drug therapy that increasing eradication reason why some report found no H. pylori in dental plaque. success rate on stomach treatment and reducing recurrence of Yang J et al.,26 reported a clinical study including 212 Han stomach H. pylori infection.29 They found the H. pylori-positive Chinese non-smoking adults. The results indicated that rate in the healthy periodontal group was 15.38%, but it was H. pylori positive status significantly increased the risk of 72.73% in periodontitis group was. They consider the dental periodontal diseases. Following is a summary table that listed plaque can be one of the main causes of recurrence infection of all articles published from 1995 to April 2016 that indicated a stomach H. pylori infection. The oral H. pylori can also be the close association of oral H. pylori and periodontal diseases in source of oral–oral transmission.30 Conclusively, a new strategy various countries with a data of a total of 61 299 individuals which concomitant eradication in oral and gastric infection can (Table 2). result in clearance of H. pylori infection.31,32 Further reports In recurrent aphthous stomatitis, there was a strong relation- found same strain of H. pylori simultaneously exists in plaque ship between oral H. pylori infection and stomach infection. and gastric mucosa. There was a positive correlation between Also, H. pylori may play an etiological role.27 An article the collected indices and quantity of H. pylori colonization.33 indicated oral H. pylori may be associated with leukoplakia There might be a relation between oral of H. pylori and oral and lichen planus oral lesions.28 lesions. Therefore, they suggested that we should have an early Since oral H. pylori infection has associated with stomach detection and eradication of oral H. pylori, especially important diseases, they reported periodontal treatment may have positive in high-risk patients.34–37 Cellini et al. found H. pylori exist in

Table 2 All articles published from 1995 to 2016 regarding the discussion on periodontal disease associated with oral H. pylori

Authors Type Country No Methods Conclusion

Nisha et al.8 Original Research India 500 Seropositive Periodontal close to H. pylori Ren et al.15 Original Research China 691 PCR Periodontal close to H. pylori Yang et al.26 Original Research China 212 PCR Periodontal close to H. pylori Gulseren et al.27 Original Research Turkey 81 RUT Periodontal close to H. pylori Kazanowska et al.28 Original Research Poland 126 PCR Periodontal close to H. pylori Zheng and Zou18 Original Research China 70 PCR Periodontal close to H. pylori Veiga et al.20 Original Research Portugal 447 PCR No close relation Ding et al.12 Original Research China 1050 HPS Periodontal close to H. pylori Ogaya et al.11 Original Research Japan 40 PCR Periodontal close to H. pylori Lauritano et al.29 Original Research Italy PCR Periodontal close to H. pylori Amin et al.31 Original Research Iran 45 PCR Periodontal close to H. pylori Anand et al.46 Review Article India PCR Periodontal close to H. pylori Adler et al.13 Review Article Argentina PCR Periodontal close to H. pylori Abadi et al.32 Original Research Iran 134 PCR Periodontal close to H. pylori Yang et al.33 Review Article China PCR Periodontal close to H. pylori Bharath et al.34 Original Research India 56 PCR Periodontal close to H. pylori Al Sayed et al.16 Review Article India PCR Periodontal close to H. pylori Boylan et al.36 Original Research USA 49120 Period exam Periodontal close to H. pylori Marbaix S et al.14 Review Article France PCR Periodontal close to H. pylori Irani et al.35 Original Research Iran 228 PCR Periodontal close to H. pylori Hirsch et al.10 Original Research Germany 10 PCR Periodontal close to H. pylori Salazar et al.37 Original Research USA 131 Seropositive Periodontal close to H. pylori Fernandez et al.7 Original Research Mexico 200 Seropositive Periodontal close to H. pylori Tsami et al.9 Original Research Greece 35 Seropositive Periodontal close to H. pylori Chaudhry et al.25 Original Research Pakistan 100 PCR Periodontal close to H. pylori Chen et al.30 Original Research China 173 Saliva HP test Periodontal close to H. pylori Navabi et al.17 Review Article Iran 1861 PCR Periodontal close to H. pylori Bago et al.39 Original Research Croatia 56 PCR Periodontal close to H. pylori Namiot et al.23 Original Research Poland 155 HP antigen No close relation Silva et al.24 Original Research Brazil 115 PCR Periodontal close to H. pylori Cellini et al.38 Original Research Italy 19 PCR Periodontal close to H. pylori Dye et al.6 Original Research USA 4504 Seropositive Periodontal close to H. pylori Butt et al.21 Original Research Pakistan 178 CLO test Periodontal close to H. pylori Hardo et al.19 Original Research UK 62 PCR Periodontal close to H. pylori

Experimental & Molecular Medicine Oral H. pylori JKC Yee

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the esophagus and human saliva sample. They suggest that and recurrence of stomach infection. Morales–Espinosa et al.48 saliva and the esophagus may be a source of stomach H. pylori report that in Mexico, many patients have H. pylori in the oral infection.38 Furthermore, Bago et al.,39 reported that almost cavity suffer from gastric symptoms. Therefore, they suggest we half of the patients suffer from gastric H. pylori harbored the should have the combination of treatments on both sites same bacterium in the oral cavity. After the eradication therapy immediately. of stomach infection, H. pylori was not detected in the oral There are several reports indicated eliminating dental plaque cavity, they proposed high effectiveness of the therapy protocol can significantly improving H. pylori of the gastric mucosa.41 in the oral cavity may not help stomach recover. They consider Zaric S et al.49, report a combination periodontal treatment and oral H. pylori as a transient character. However, Bago et al.39 is drug systemic therapy can increasing the eradication success only one study that shows oral H. pylori was clean after rate of stomach H. pylori infection and decreasing the risk of eradication on the stomach, which did not agree with all recurrence of stomach infection. Therefore they suggested that remaining studies on this subject in past 20 years. we should do professional plaque removal and oral hygiene procedures along with the antibiotic treatment of stomach THE ASSOCIATION BETWEEN STOMACH AND ORAL H. pylori. infection.50,51 Since the cagA gene exists in both H. PYLORI INFECTION gastric biopsies and saliva, as well as dental plaque52 this Whether the oral H. pylori are transient or permanent in the evidence further supports the view of a close association mouth, the fundamental question is, ‘Can oral H. pylori be a relationship between oral and stomach H. pylori infection. Al reservoir for gastric H. pylori infection?’ One might be first Asqah et al.33 conducted a clinical study that shows 65% of address whether there is an association between oral and gastric patients have dental plaque H. pylori. Among them, there were H. pylori carriage. Several studies have reported there is positive 450% patients’ harbored same bacteria in their stomach. In – link oral with gastric H. pylori.40 42 Conversely, there was a the same manner, the periodontitis patients had a significantly study that indicated there were no such association.43 Song higher percentage of H. pylori in their dental plaque and the et al.44 reported that H. pylori exist in the oral cavity of 97% of stomach that compared with patients without periodontitis. In patients that has characteristic distribution independent of the addition, 78% of patients have H. pylori dental plaque in status of stomach infection. This is why they consider H. pylori periodontitis group versus only 30% in non-periodontitis may belong to the normal oral microflora, which is nothing group. The coexistence of H. pylori in both dental plaque link with stomach infection. However, recent studies show the and the stomach had been observed.53 Liu Y et al.54 found bacterial involving oral cavity and stomach has an identical or dyspeptic patients with gastric infection are more likely to closely species which related strains of H. pylori that provided a harbor H. pylori in their mouth that show a close association good evidence of the role of the oral cavity link with gastric between H. pylori in the oral cavity and the stomach. Loster infection. et al.55 found an interesting relationship of the lengths of One of the articles has a view against oral H. pylori link with dentist occupations. The dentist may carry gingival sulcus stomach infection because they found oral and stomach infection with H. pylori after a long time working on a patient H. pylori have different genotypes. This study shows that more with oral H. pylori infection that indicating oral H. pylori can be than one H. pylori strain exists in the oropharynx and stomach contagious through dental instruments. Yee et al.56,57 had at the same patient. They concluded that oropharyngeal conducted several large clinical trials in China where there were infection is independent of the gastric infection.10 However, 410 000 individuals involved. More authors in addition to Yee remarkable genotype diversity among stomach, saliva and stool et al.56,57 conducted similar clinical trial in various countries. that showed that more than one H. pylori genotype may exist in All of them found a strong association of oral and stomach H. the same patient.45 However, there is an article reported the pylori infection Table 3. same strain of H. pylori simultaneously exists in plaque and However, Silva et al.58 had different viewpoints. Because they gastric mucosa.34 cannot found H. pylori in any oral samples who suffer from There is increasing evidence recently regarding the role of stomach H. pylori infection. Also, they found no genotype cagA the oral cavity in the transmission of H. pylori to stomach use in oral samples and cannot characterize vacA genotype in an new methods to detecting H. pylori in the oral cavity. That oral sample of 430 patients. evidence continually supporting the view of the association between oral and stomach H. pylori infection. But, Young WHY THE TRADITIONAL DRUG ERADICATION OF et al.46 Consider this subject requires considerably more clinical GASTRIC H. PYLORI INFECTION IS INEFFECTIVE studies before make a definite conclusion; especially we need a AGAINST ORAL H. PYLORI INFECTION? technology to confirmed oral H. pylori exists. As long as we can In 1999, Dore-Davin et al.59 first discovered after systematical confirm oral cavity involving, then we can do preventive eradicationonstomachH. pylori infection that had no effect on measures oral transmission. Rasmussen et al.,47 report a strong oral H. pylori.Miyabayashiet al.60 further reported the link between oral H. pylori and gastric infection in Brazilian eradication success rate of stomach H. pylori infection was community. In their finding, the H. pylori exist in the oral significantly lower in the oral H. pylori-positive cases compared cavity with different distribution between saliva and dental with oral H. pylori-negative cases at 4 weeks after drug plaques that may suggest a potential link between oral infection treatment. Two years later, they found 95.8% of patient had

Experimental & Molecular Medicine Oral H. pylori JKC Yee

5 no stomach H. pylori infection with oral H. pylori-negative stomach H. pylori infection of dyspeptic patients. After half cases but they found only 69.5% of patient with no stomach year, the stomach H. pylori infection of the group received H. pylori infection with oral H. pylori-positive. They concluded periodontal treatment has much lower than the group received that oral H. pylori infection affected eradication successes rate no periodontal treatment. However, our studies1 shown that and oral H. pylori infection has a strong link with a recurrence patients who received teeth cleaning had no effectiveness on H. of gastric infection.60 Since 1999, there are numerous studies pylori infection of mouth by statistical analysis. The special that show when patients received drug treatment on stomach mouth resin is best effective in eliminating oral H. pylori – H. pylori infection that cannot clean up oral H. pylor.1,32,39,61 64 infection that I will discussion it at the end. All later reports support Dore-Davin’s first discovery (Table 4). The reason why eradication on stomach H. pylori infection In terms of how to treat an oral H. pylori infection, there are has no effective on H. pylori infection of the mouth, because a number of studies showing that mouth rinse treatment alone H. pylori exist in between the teeth and gums called, an area or combined with periodontal therapy may eliminate oral referred to as the ‘bio- film membrane’ (Biofilm), also we H. pylori infection and increase the eradication success rate of called as plaque barrier. The drug cannot penetrate it when the stomach H. pylori infection.1,2,63 A clinical study showed that patient received symmetrically eradication. This is why the the symptoms of H. pylori-positive dyspeptic patients may conventional treatment on stomach H. pylori infection had no – improve by oral treatmet.2 There are three studies65 67 that effect on oral H. pylori; especially it exists in dental plaque. evaluated the effectiveness of periodontal treatment on H. pylori of mouth. They reported that patient received THE ERADICATION OF STOMACH H. PYLORI INFECTION periodontal treatment may decrease plaque H. pylori. After FACES MORE CHALLENGING THAN EVER DUE TO treatment, if those patients still suffer from plaque H. pylori PROGRESSIVE LOSS EFFICACY OF TRADITIONAL then followed a combination of treatment with triple therapy. THERAPY Jia et al.67 proposal a periodontal treatment before eradication There were several proposals how to providing treatment after on stomach H. pylori infection. They reported that the prior failure of second-line therapies. One of them is the endoscopic- periodontal intervention significant increasing eradication of guided antibiotic susceptibility testing. However, according to

Table 3 Studies show the association of oral and stomach H. pylori infection

Author Country No Method Conclusion

Yee et al. 20 China 410 000 HPS Association of Oral and stomach H. pylori Medina et al.51 Argentina 8 PCR Association of Oral and stomach H. pylori Eskandari et al.50 Iran 67 PCR Rasmussen et al.47 Brazil 78 PCR Association of Oral and stomach H. pylori Loster et al.55 Poland 46 dentists Serological test Association of Oral and stomach H. pylori Liu et al.54 China 443 PCR Association of Oral and stomach H. pylori Al Asqah et al.53 Saudi Arab 101 Urease test Association of Oral and stomach H. pylori Silva et al.58 Brazil 62 PCR Association of Oral and stomach H. pylori Zaric et al.49 Serbia PCR Association of Oral and stomach H. pylori Jia et al.67 China 56 PCR Association of Oral and stomach H. pylori Morales et al.48 Mexico 65 PCR Association of Oral and stomach H. pylori Oshowo et al.41 UK 208 PCR Association of Oral and stomach H. pylori Maplstone et al.40 UK 13 Nested PCR Association of Oral and stomach H. pylori

Table 4 Eradication on stomach H. pylori infection had no effects on oral H. pylori

Author Country No Time after eradication Positive H. pylori in Oral Positive H. pylori in Stomach

Adadi et al.32 Iran 132 After eradication Patients carrying H. pylori Patients carrying H. pylori Wang et al.1 China 159 4 weeks 49.44% 38.6% Song and Li63 China 391 4 weeks 33.2% 21.6% Bago et al.39 Croatia 56 3 months 0 21.7% Gao et al.62 China 96 4 weeks 62.8% 32.4% Zaric et al.49 Serbia 52% 23% Gebara et al.61 Brazil 30 3 months 60% 10% Miyabayashi et al.60 Japan 47 4 weeks and 2 years 69.5% 4.2% Dore-Davis et al.59 Swiss 22 4 weeks 57%

Experimental & Molecular Medicine Oral H. pylori JKC Yee

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the principal of Maastricht Guidelines, its role has expanded infection. A new strategy that concomitantly eradicates oral and over, over again in past years. Several authors have reported the gastric colonization would result in clearance of H. pylori results of such proposal. The developed both efficacy clinical infection and improve the eradication rate of gastric H. pylori.1 trials and cost-effectiveness trials against drug-resistant of The discovery of oral H. pylori is especially significant and treatment on stomach H. pylori infections. However, their meaningful because this motor system can explain why the results are not very successful, because antibiotic resistance is recurrence of stomach H. pylori infection occurs. not the only main reason for the failure so far. The failure becomes a good attention in medical societies worldwide, UREA BREATH TEST, A GOLD STANDARD DIAGNOSIS, special in Asia.68 It is time now we should establish a new USE ONLY FOR DIAGNOSIS OF STOMACH H. PYLORI view besides antibiotic resistance, which are the most impor- INFECTION tant issues for the progressive loss of efficacy of eradication due Urea breath test (UBT) C13 is a trusted diagnostic procedure to oral facts. Yee69 proposed the key conception that H. pylori used to identify stomach infections by H. pylori72 with the have a second colonized site in an oral cavity in addition to the exception of a small number of false positives73 and they stomach. However, some authors said ‘oral H. pylori cannot be diagnosis for all H. pylori species not specific for CagA.74 The cultured’, ‘H. pylori exists in the oral cavity are dead bacterial principle is based upon H. pylori to transform urea that that has no effect on stomach treatment’ and ‘the oral cavity is released by H. pylori to carbon dioxide and ammonia. UBT not a colonized site,’ which has become the main reasons to is a popular method for diagnosis of H. pylori of the stomach. deny our version of oral H. pylori colonization. Because the It holds efficacy at 96.7% sensitivity and 96.2% specificity. majority of physicians working in gastroenterology field ignore However, UBT is not used for diagnosis of oral H. pylori the oral H. pylori, ~ 20% of the population of Asia suffers from because C13 or C14 are not dissolved in the mouth during the 1,3 70 oral H. pylori infection . Not only in Asia, Jonaitis et al. testing. In medical practice, doctor considers you have no reported that after H. pylori eradication they frequency observe stomach H. pylori infection if you have negative results of UBT on H. pylori recurrence of peptic ulcer patients during 9 years C13. In fact,doctor only pays attention to stomach infection. In in Lithuania The recurrence rate of H. pylori is high at 27.2%. their view, there is no H. pylori infection exist in anywhere This number is very close that we found in Asia. besides stomach. This is traditional view on H. pylori infection There is a motor circulatory system that I summarized that for many years. However, the clinical study provides evidence behind the negative impact of oral H. pylori on stomach that shown H. pylori oral infection are nonetheless present that treatment (Figure 1). This system contains two colonized sites also negatively effective on eradication on stomach H. pylori of H. pylori in the upper digestive system. The primarily infection. In Asia, approximately 20–30% of the population colonized site resides in the stomach and delivers H. pylori into having oral H. pylori infection but with negative UBT results.1 the oral cavity by reflux as a conveyor. The oral cavity is a We developed a technology, H. pylori saliva test (HPS) that second colonized site for the culture of H. pylori; bacteria from especially detecting oral H. pylori4 and it is non-invasive, fast this site drop into the stomach by the swallow reflex as a result and no equipment required during the testing. conveyor. Two colonized sites with two conveyors have been It is controversial the fact of H. pylori exists in the oral cavity constructed to create a system that transports H. pylori along in past 20 years. It divided scientists into two groups. The the upper digestive system. One of the colonized sites contains majority scientism belongs to the first group that proposed that H. pylori, which results in a second colonized site being H. pylori stay in the oral cavity and that all positive results occupied by H. pylori. The recurrence of H. pylori infection fl occurs if one site had been treated by a drug and another site detected by PCR are fragments of dead bacteria that re ux from the stomach which could not be cultivated from PCR- had not. The motor circulating system can transport H. pylori 75 along the circle. A number of studies have shown that oral positive samples. The proposal says the oral H. pylori come fl H. pylori were not eliminated in patients who received a drug from stomach re ux was survive only a few hours in the treatment for stomach H. pylori.13,63,71 Traditional drug mouth. Because oral cavity holds high oxygen concentration eradication and teeth cleaning had an effectiveness rate of less that kills all H. pylori. If the proposed view is correct, then the than 10%. By statistical analysis, there was no effect all on oral fragmented of bacteria have no negative effect on eradication of stomach H. pylori infections.75,76 However, their view contra- dicts with the studies of PCR recently published,77,78 the fact of the oral cavity may have hypoxia environment,35,79 the fact of oral H. pylori infection cannot clean up by traditional therapy.32,46 The fact that indicated H. pylori can be cultured from saliva sample if we use a new method of culture.1 The fact of oral and stomach has the same gene of H. pylori.7,55 It also contradicts with the evidence of the lower rate of eradication of stomach H. pylori when a person suffers from oral H. pylori infection41 and the fact of results of all meta-analysis published Figure 1 A motor system between two colonized sites. in the past.13,71 Therefore, we proposed a new view that

Experimental & Molecular Medicine Oral H. pylori JKC Yee

7 indicated H. pylori colonization of the oral cavity which may word he did not trust our new technology, HPS can detect oral resolve all previous issues in past. H. pylori. If a patient with H. pylori infection of the stomach, then it is WHAT IS THE BEST TEST FOR DETECTING H. PYLORI a good reason to believe the oral bacterial come from stomach OF THE ORAL CAVITY IN CLINICAL SETTINGS? reflux. But, with a UBT-negative patient and negative culture of PCR is a method often used for detecting oral H. pylori, but its stomach sample, we still detected H. pylori in the mouth. Also, results have high variation. Some articles report the detecting we observe a large number of the patient which near 10 000 rate were zero, but some articles report with 90% of positive patients with negative UBT test in several clinical studies but response on the oral sample. Naturally, the scientificcommu- see oral H. pylori. This is why we confidence our data are nity very confused on the large variation of PCR testing results. reliable. We should find out why such discrepancies exist.22 In term of If we confirmed H. pylori-colonized in the oral cavity by the requirement of the sample, expensive equipment required culture saliva sample? What will be our next step? To follow up, and technician for operating PCR testing. So PCR method is we have conducted several studies in several subjects (1) What not a good and convenient way to detecting oral H. pylori for is the recurrence rate of stomach H. pylori infection each year clinical settings. Therefore, a diagnostic method has a high to oral H. pylori? (2) Is it true that systematically drug sensitivity and specificity for oral sample should be established. treatments are not effective on oral H. pylori due to the We believe that HPS is a good and convenient test for diagnosis construction of dental plaque structure? And (3) is the of H. pylori in the oral cavity. As long as we have an easy, eradication rate of stomach H. pylori infection getting lower accrue test, then the clinical trial can be carried out on a large each treatment? number of patients to obtain a good size of clinical data, which will help to understand the strong links between oral and CULTURE OF ORAL H. PYLORI stomach H. pylori infection. We are able to establish the Krajden et al.80 in 1989 first reported the result of the culture of principles of evidence regarding oral H. pylori infection. H. pylori gastritis. There was only one plaque had a positive HPS is a lateral flow immunochromatographic test device result among seventy-one patients. All seventy-one saliva that uses saliva as testing sample detecting oral H. pylori within cultures show negative result. Since then, many attempted to few minutes. The principle of HPS is based on monoclonal cultivate oral H. pylori had been rarely successful – antibody react with oral urease produced by H. pylori. worldwide (Table 5).19,30,53,81 107 Indeed, published articles A laboratory study was conducted to determine its specifi- reported culture-positive rates are very low from various oral city. The following common bacteria of oral cavity were sample included saliva, dental plaque, and teeth. Majmudar applied: Streptococcus gordonii, S. mutans (major pathogen of et al.,97 D'Alessandro and Seri,102 reported that they had been dental caries), S. salivarius, S. sanguinis and , successfully cultured on saliva; however, Namavar et al.101 (major pathogen of periodontitis), consider their results were false positives. The main difficulties Gemella haemolysans, Granulicatella adiacens, of bacteria culture of the oral sample; are how to collect oral rectus (major pathogen of periodontitis, species related to specimen; how to preserve it; there was a small number of Helicobacter), Corynebacterium matruchotii, Bifidobacterium colonies of H. pylori for culture and competition with other dentium, Actinomyces naeslundii, A. odontolyticus. All the above oral bacteria. Because the concentration of H. pylori of the bacteria did not show interference or cross-reactivity with stomach is three magnitudes higher than that of the oral cavity – HPS test. (105 CFU per ml versus102 CFUper ml108 110), it would be The sensitivity of the tests was 10 ng ml − 1 H. pylori antigen.4 insufficient to use conventional stomach culturing techniques for detecting oral H. pylori. The method must be adapted to DO ORAL H. PYLORI COME FROM STOMACH REFLUX? obtain a high positive rate of oral H. pylori culture with very Do H. pylori in oral cavity come from stomach? That is one of low concentration of the oral sample. However, Dowsett the key issues that had been disputed in past. The author had a et al.111 dispute that If such low concentration of H. pylori in discussion on this question with the Nobel Laureate, Dr. Robin the oral then it will be an insufficient number of bacterial result Warren. He indicated that oral H. pylori have to have come infection in any where. If this is indeed the case, the subsequent from the stomach through the reflux motion. In the case, if the ability of oral H. pylori has negative on stomach infection that patient had no H. pylori of the stomach, then why oral H. pylori may be questionable. exist? He believes very low numbers of bacteria lives in the Author’slabhadbeencultureH. pylori on saliva successfully stomach, but show false-negative UBT tests. They can through since 2012.H.Pyloriwere isolated from saliva by pretreatment reflux the bacteria back to the oral cavity. Since scientist cannot with Urea-Hydrochloric acid.108 We performed all the follow- culture on a positive sample by PCR that leading him to be ing tests to confirm H. pylori colonization:(a) Oxidase test (b) trusted there are no living H. pylori in the oral cavity. His past Catalase test (c) H. pylori antigen and antibody test (d) lab experience also shows that he never managed to culture H. Microscopy observations. pylori from food, water or the mouth. He also advice when we I then communicated with Dr. Floyd E. Dewhirst, a say antigens of saliva or plaque, we should very carefully tell significant scientist related to this field112 regarding confirma- what methodology that to demonstrate those antigens. Another tion on the result of H. pylori culture. He indicated that this is

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Table 5 Articles published since 1989 regarding culture of colonizes the GI tract. Organic bacteria are very specificintheir H. pylori in oral cavity niche selection. There are nine niches in the oral cavity—sub- gingival, supra-gingival, tongue, tonsils, throat, attached gin- Dental giva, cheek, palate and saliva—and each niche has a distinct Author Country N0 plaque Saliva Teeth microbial (some overlap). The oral cavity has no site with Krajden et al.80 Canada 71 1 0 gastric mucus and a pH of close to 1. Where do we think it Oshowo et al.51 UK 180 2 0 colonizes? Dr Dewhirst examined 27 subjects at the nine oral Cheng et al.30 UK 122 0 0 1 niches and reviewed the site sample reads by illuminate 16S Luman et al.82 UK 109 0 0 0 rRNA of the V1–V3 region. The region had about 100 000 Allaker et al.83 UK 100 0 0 0 reads per site, so 100 reads are 0.1% of the total, plus he never Bernander et al.84 Sweden 114 0 0 0 saw H. pylori in any subject, at any site. Maybe his subjects did Pustorino et al.85 Italy 83 5 0 0 not have H. pylori in their stomach—this was not the point of Khandaker et al.86 UK 81 12 0 0 the study. However, if we believe it is part of the microbiome in Ishihara et al.87 Japan 82 0 0 0 the mouth, then we need to show where in the mouth and at Hardo et al.19 UK 62 0 0 what percent of the population. If we find it at a high 88 Majmudar et al. India 40 40 concentration in some oral niche, then fine—we can say we 89 Cellini et al. Italy 31 1 see it in at least one person at that particular concentration in 90 Wahlfors et al. Finland 29 0 0 this site/niche. However, this still does not address the question 42 Paronnet et al. USA 26 3 of transient versus commensal (or indigenous microflora). Namavar et al.91 Holland 20 1 However, the author considered the clinical trial of eliminating D'Alessandro and Seri92 Italy 20 16 H. pylori of the oral cavity which increase the successful rate of Ferguson et al.93 USA 16 1 eradication of stomach H. pylori infection, and the best Bickley et al.94 UK 15 0 Zhent et al.30 China 72 31 evidence of H. pylori in the oral cavity is commensal fl 113 Chen et al.30 China 173 69 micro ora. 96 Zheng et al. China 163 42 H. PYLORI, A Jiang et al.97 China 50 13 ARE SEXUALLY TRANSMITTED Xu et al.98 China 98 21 BACTERIUM? Agarwal et al.99 India 30 9 If the view and evidence of H. pylori-colonized in the oral Czesnikiewicz-Guzik et al.100 Poland 100 45 55 cavity are correct, then oral H. pylori can be sexually Czesnikiewicz-Guzik et al.101 Poland 100 46 54 transmitted bacteria. On the other hand, if no H. pylori Loster et al.102 Poland 46 22 20 sexually transmitted diseases through oral exist, how we can Sudhakar et al.103 India 50 10 believe that H. pylori exist in the oral cavity? Let us check the Teoman al.104 Turkey 67 17 reference here to see what they reveal it. Umeda et al.105 Japan 57 18 Use blood H. pylori test run a clinical study on sex partners Goosen et al.106 South Africa 58 2 with man and woman. The results show H pylori, the non- Majmudar et al.88 India 40 40 infected individual had statistically significant different pre- D'Alessandro et al.92 Dell’ Aquila 20 16 valence rates of sex diseases. There were 83.3% vs 28.5%, Namavar et al.91 Netherlands 20 3 respectively,113,114 which may indicate H pylori may be a sex Me'graud et al.122 transmitted bacterium. Wang et al.1 China 159 94 Several studies have shown there is a strong relationship between sexually transmitted disease and ethnicity minorities’ groups.115,116 They report the high rates of sexually transmitted not sufficient; we should have a full 1500 base 16S rRNA infection occur in ethnic minorities. 114 sequence of the isolate(s), and deposit the organisms in a Schutze et al. reported the H. pylori infection are national culture collection. With sequences and strains, the contingent and transmitted between spouses. They found a fi work is much stronger. He suggested the existing published recurrence of H. pylori spouses contains same identi ed strain studies show H. pylori can be cultured19,30,53,81–107 were not gene type from spouses which is a good evidence that indicated accountable because of none of them involved work with a full H. pylori transmitted among spouses. However, they also found multiple strains exist in the same individual. 1500 base 163r RNA sequence. He further indicated that the There were very limited articles published regarding sexual confirmation of culture results do not address transient (from transmission of H. pylori among female sex workers. Eslick burp) versus colonization. The oral microbiome and the gut GD117 reported that so far even there is no study conducted a microbiome each have greater than 700 species. The number of prevalence of H pylori infection among female sex workers. species shared between these two different habitats is one By theoretical analysis, they proposal H. pylori may colonize organism (Dialister pneumosintes). Even though vast quantities in the vaginal associated with yeast which constructed as of oral bacteria are swallowed every day, and none (except one) biofilm formation, based on H. pylori exists in the biofilms

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9 among many bacterial species. This is why treatment failure proportion of males suffers with non-gonococcal urethritis, but occurs on yeast infection of the vaginal site. Eslick further no responsible bacteria found? This was an article suggested we hypothesized that H. pylori may colonize in an acidic vaginal should link urethritis with H. pylori infection and urethritis.124 environment that making vaginal as a source for sexual When I have a personal conversation with Dr Kast RE who transmission for many species of bacteria.118 hypnosis on the link between prostate and H. pylori infection It was interesting an early case report that said they found because fellatio was so popular. ‘spiral bacteria’ with same strains of H. pylori in a woman’s An article reported vaginal yeasts as primary reservoir of vagina who suffers from vaginitis. They describe spiral bacteria H. pylori that may facilitate transmitted it to neonates. Mother have comma-shaped rods (1–4 μm in length) with a corkscrew with UBT positive may transmit H. pylori through vaginal motion and its head bear with four to eight flagellae. This delivery to neonates based on there are close associated oral finding was a year before original article of Warren and yeasts of neonates and vaginal yeasts.125 Marshall that discovered on stomach H. pylori infection. Healthcare workers special baby delivery workers are more Besides their appearance was similar, they also found that careful on H. pylori infection because it is contagious during some of those spirals bacteria can be cultured under micro- vaginal delivery procedures. aerophilic condition after 72 h incubation at 37 °C. The An article indicated H. pylori may transmit through fellatio biochemical profile was very similar between spiral bacterial in the urethra. However, they proposal further research is and H. pylori, but they did not run a further test to confirmed required to defined the link between in H. pylori and both of them are same species. Several articles indicated vertical urethritis.126 transmission of H. pylori exists in the vagina during the birth process. The prevalence of H. pylori in pregnant women is PREVENTION ORAL–ORAL TRANSMISSION OF about 20%.119,120 H. PYLORI Kast RE reports a case that oral directly contacts with the Chow et al.,127 found a strong association between prevalence nipple that may result in the retrograde propulsion of H. pylori of H. pylori infection and chopsticks user in Chinese society of into breast ducts which may lead to fibrocystic breast changes. Australia. They consider the saliva containers with H. pylori It is a heterogeneous group of benign. In this case report, the through chopsticks. woman had an H. pylori serology diagnosis as negative. On the basis of the fact of oral to oral transmission, water However, after antibiotic eradication, she had no more pain carry and fecal to oral transmission, Dowsett et al. indicated and tenderness in breast and her breasts normalized.121 This H. pylori infection have been population dependent.128 A was the reason leading his hypnosis that this woman had report from India that indicated there was a significant H. pylori retrograde into breast duct results H. pylori local association between H. pylori infection and fingernail carriage infection. An article reported that mothers had been diagnosis based on H. pylori had been detected by PCR on beneath of with H. pylori antigenuria, the fecal of their half of breastfed fingernails. It is a customer of eating way in India. They use a 3 days old neonates found H. pylori.122 Are H. pylori finger to hold food. So washing hands before eating may be transmitted from mother to neonates? Or from vertical important for stop H. pylori’stransmission. transmission through vaginal delivery? They also found By blood H. pylori antibody study, they found a strong link H. pylori exist in 4 out of 66 milk samples of mother suffered between H. pylori infection and crowded living condition.128 from H. pylori infection.123 They further consider the socioeconomic status become an Since oral sex is a very popular worldwide toady, the action important issue that influences their finding. In developing of fellatio from woman to man, the H. pylori can transmit into countries, special in low socio-class children suffer from the urethra that resulting infection. The question is why a large H. pylori infection become an important issue. There are no symptoms at all during a long period of latency until adult. Same way as stomach cancer resulted by stomach H. pylori infection usually does not show until older age.129 Regarding H. pylori transmission, mother as H. pylori carrier may be the main source for childhood H. pylori infection.130 A review article131 reported the prevalence rates vary widely with different ethnic groups and geographical location. An oral–oral route of transmission had been mentioned with all studies that indicated a fact of H. pylori exists in the oral cavity. Dowsett and Kowolik22 dispute that if the fact of H. pylori transmission through oral to stomach, then we naturally expect oral H. pylori infection should be more often than stomach H. pylori infection. Our studies have shown the discovery of oral H. pylori is especially significant and meaningful that may Figure 2 The accuracy of HPS confirmation by S-HP-C. answer the question raised by Dowsett and Kowolik, because it

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is ~ 20% of the population of Asia suffers from oral H. pylori infection. 1 Wang XM, Yee KC, Hazeki-Taylor N, Li J, Fu HY, Huang ML. Oral , its relationship to successful eradication of gastric H. pylori and saliva culture confirmation. J Physiol Pharmacol NON-ANTIBIOTIC TREATMENT FOR ELIMINATING ORAL 2014; 65:559–566. H. PYLORI 2 Anand PS, Kamath KP, Anil S. Role of dental plaque, saliva and There is a non-antibiotic treatment for oral H. pylori infection periodontal disease in Helicobacter pylori infection. World J Gastroenterol 2014; 20:5639–5653. 1 available. Our studies indicated e-polylysine (L) and the 3 Sheu BS, Cheng HC, Yang YJ, Yang HP, Wu JJ. The presence of dental Glycerol Monolaurate (GM) may eliminate oral H. pylori. disease can be a risk factor for recurrent Helicobacter pylori infection after eradication therapy: a 3year follow-up. Endoscopy 2007; 39:942–947. L L ‘ ’ The holds around 30 -lysine residues. They use e as a link 4 Yee KC, Wei MH, Yee HC, Everett KD, Yee HP, Hazeki-Taylor N. with all Lysine molecules. Its surface has cationic. From A screening trial of Helicobacter pylori-specific antigen tests in saliva to electronically point view, the surface of H. pylori charges with identify an oral infection. Digestion 2013; 87:163–169. 5 Huang R, Ding P, Huang D, Yang F. Antibiotic pollution threatens public positive power. The lysine amino acids are molecularly linked health in China. Lancet 2015; 385:773–774. by the epsilon amino group and the carboxyl. If lysine meets 6 Dye BA, Kruszon-Moran D, McQuillan G. The relationship between periodontal disease attributes and Helicobacter pylori infection among with H. pylori in water, they will be having the very strong adults in the United States. Am J Public Health 2002; 92:1809–1815. electronically huge power that making a cell of H. pylori 7 Fernández-Tilapa G, Axinecuilteco-Hilera J, Giono-Cerezo S, Giono-Cerezo S, attached with lysine molecules. The many liners of lysine Dinorah-Nashely MC, Illades-Aguiar B. vacA genotypes in oral cavity and Helicobacter pylori seropositivity among adults without dyspepsia. Med Oral molecules have clockwise and counter clockwise motion like a PatolOralCirBucal2011; 16:e175–e180. knife that tear membrane of H. pylori as fragments. The oral 8 Nisha KJ, Nandakumar K, Shenoy KT, Janam P. Periodontal disease and Helicobacter pylori infection: a community-based study using serology and H. pylori will die. rapid urease test. J Investing Clin Dent 2016; 7:37–45. We found GM exist in mother’s milk that is nature 9 Tsami A, Petropoulou P, Kafritsa Y, Mentis YA, Roma-Giannikou E. The preventive material for human's milk that formed by glycerol presence of Helicobacter pylori in dental plaque of children and their parents: is it related to their periodontal status and oral hygiene? Eur J and lauric acid. In the water, GM will kill H. pylori immedi- Paediatr Dent 2011; 12:225–230. ately. We use Lysine and GM, called L-GM formed in mouth 10 Lukeš P, Pavlík E, Potužníková B, Plzák J, Nártová E, DosedělJet al. washing solution treat oral H. pylori infection. The patient uses Comparison of Helicobacter pylori genotypes obtained from the oropharynx and stomach of the same individuals - a pilot study. Prague Med Rep it twice a day and 20 cc each time with 5 min in the mouth. 2012; 113:231–239. After 2 months, we found increasing efficacy of eradication on 11 Ogaya Y, Nomura R, Watanabe Y, Nakano K. Detection of Helicobacter fl 1 pylori DNA in in amed dental pulp specimens from Japanese children and stomach infection about 21%. These results of improvements adolescents. J Med Microbiol 2015; 64(Pt 1): 117–123. had been confirmed by Saliva H. pylori culture (S-HP-C; 12 Ding YJ, Yan TL, Hu XL, Liu JH, Yu CH, Li YM. Association of salivary Figure 2). On the basis of the confirmation of S-HP-C, we Helicobacter pylori infection with oral diseases: a cross-sectional study in a Chinese population. Int J Med Sci 2015; 12:742–747. calculated the sensitivity, specificity, accuracy and positive and 13 Adler I, Muiño A, Aguas S, Harada L, Diaz M, Lence A. Helicobacter pylori negative predictive values of HPS as 98.1, 94.1, 96.5, 96.2 and and oral pathology: relationship with the gastric infection. World J Gastroenterol 2014; 20:9922–9935. 97% respectively. 14 Marbaix S, Soueidan1 A, Romani M, Campard G, Amador G, Badran Z. Helicobacter Pylori and periodontal diseases: an update and proposal of a CONCLUSION multidisciplinary clinical protocol. Open J Stom 2013; 3:318–322. 15 Ren Q, Yan X, Zhou Y, Li WX. Periodontal therapy as adjunctive treatment A colonized site of H. pylori can exist in the oral cavity. for gastric Helicobacter pylori infection. Cochrane Database Syst Rev In medical practice, doctor consider patients had no stomach 2016; 2: CD009477. H. pylori infection. But in fact, patients can have negative 16 Sayed A, Anand PS, Kamath KP, Patil S, Preethanath RS, Anil S. Oral cavity as an extragastric reservoir of Helicobacter pylori. ISRN Gastro- results of UBT, but H. pylori exist in oral cavity. UBT cannot enterol 2014; 26: 1369. detect oral H. pylori. 17 Navabi N, Aramon M, Mirzazadeh A. Does the presence of the Helicobacter If there is a live H. pylori colony in the oral cavity, then it pylori in the dental plaque associate with its gastric infection? A meta- analysis and systematic review. Dent Res J (Isfahan) 2011; 8: 178–182. would have a negative influence on the eradication of a 18 Zheng P, Zhou W. Relation between periodontitis and Helicobacter pylori stomach infection. In the classic H. pylori eradication pro- infection. Int J. Clin Exp Med 2015; 8: 16741–16744. 19 Hardo PG, Tugnait A, Hassan F, Lynch DA, West AP, Mapstone NP et al. grams, there are no clear measures of oral H. pylori; frequent Helicobacter pylori infection and dental care. Gut 1995; 37:44–46. relapses become more critical. 20 Veiga N, Pereira C, Resende C, Amaral O, Ferreira M, Nelas P et al. Oral and gastric Helicobacter pylori: effects and associations. PLoS ONE 2015; 10: e0126923. CONFLICT OF INTEREST 21 Butt AK, Khan AA, Bedi R. Helicobacter pylori in dental plaque of Theauthordeclaresnoconflict of interest. Pakistanis. J Int Acad Periodontal 1999; 78:78–82. 22 Dowsett SA, Kowolik MJ. Oral Helicobacter pylori, can we stomach it? Crit – ACKNOWLEDGEMENTS Rev Oral Biol Med 2003; 14:226 233. 23 Namiot DB, Leszczyńska K, Namiot Z, Chilewicz M, Bucki R, Kemona A. I thank Dr Qilong Yi for statistical analyses and Bolina Long for figures The occurrence of Helicobacter pylori antigens in dental plaque; an analysis. association with oral health status and oral hygiene practices. Adv Med Sci 2010; 55:167–171. ’ 24 Silva DG, Stevens RH, Macedo JM, Albano RM, Falabella ME, Fischer RG PUBLISHER SNOTE et al. Presence of Helicobacter pylori in supragingival dental plaque of Springer Nature remains neutral with regard to jurisdictional claims in individuals with periodontal disease and upper gastric diseases. Arch Oral published maps and institutional affiliations. Biol 2010; 55:896–901.

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